A traceless Trichoderma fungal gene editing method
A gene, Trichoderma technology, applied in the field of gene cycle editing of Trichoderma untraceable, can solve problems such as inability to explain the comprehensive results of multi-gene product interaction, complex internal mechanism, etc.
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Embodiment 1
[0044] Implementation 1: Construction of auxotrophic strain NJAU4742-Δura3 traceless mutant of Trichoderma Guizhou NJAU4742 (1) Construction of knockout fragment
[0045] In the process of seamless knockout of ura3, the presence or absence of the gene itself is used as a screening marker for secondary homologous recombination, so the construction of the knockout fragment is slightly different. The knockout fragment consists of 4 different parts: upstream fragment + downstream fragment + HygB resistance gene expression box + gene fragment. Among them, the HygB resistance gene expression cassette can be transcribed and translated into hygromycin B phosphotransferase, making the original strain NJAU4742 resistant to hygromycin B. After the second homologous recombination, the HygB resistance gene expression cassette and ura3 Gene sequences will be deleted together, so 5-FOA can be used for reverse screening to obtain a traceless knockout mutant of the ura3 gene. In summary, in t...
Embodiment 2
[0053] Implementation 2: The traceless knockout of the xyr1 gene, which regulates lignocellulosic degrading enzymes in Trichoderma Guizhou NJAU4742
[0054] Trichoderma strain: Guizhou Trichoderma NJAU4742-Δura3 (NJAU4742-Δura3)
[0055] (1) Construction of knockout fragments
[0056] Knockout fragment amplification primers are: upstream fragment amplification primer x1_upF: GGCCTTGAAACGGTATGTCGA (SEQ ID NO.11) and x1_upR: CGTACACCACCATCACAGGGATATCAATAGGAGATGGCTGAACTGTGTG (SEQ ID NO.12); downstream fragment amplification primer x1_downF: CACACAGTTCAGCCATCTCCTATTGATATCCCCTGTGATGTQGTACG 和x1_downR:GCCATATTGATGTAAGGTAGCTCTCCTCACTTCCGCTTCACATAGACC(SEQ ID NO.14);U3表达框扩增引物U3box_F:GAGAGCTACCTTACATCAATATGGCGCGCAGATGTAGCGGTACATG(SEQ ID NO.15)和U3box_R:GGTACTATGGCTTAGATGGAATACCCCGTATTCTCTGCCCTTGTTGC(SEQ ID NO.16);基因片段扩增引物x1_geneF:GGGTATTCCATCTAAGCCATAGTACCCTTCTTCAGCCCTTGATCCACAC(SEQ ID NO. 17) and x1_geneR: CCTTGATTCACACGCAAATGTTCC (SEQ ID NO. 18). The final concentration of the knockout...
Embodiment 3
[0064] Implementation 3: Trichoderma Guizhou NJAU4742 carbon source inhibition regulatory gene cre1 traceless knockout and ura3 gene complement Trichoderma strain: Guizhou Trichoderma NJAU4742-Δura3-Δxyr1 (NJAU4742-Δura3-Δxyr1)
[0065] (1) Construction of homologous knockout fragments
[0066] The knockout fragment is: cre1 gene upstream fragment + U3 expression box + cre1 gene downstream fragment. The amplification primers are: upstream fragment amplification primer c1_upF: GGTGGGCAAAAAGGAACCTGG (SEQ ID NO.21) and c1_upR: GCCATATTGATGTAAGGTAGCTCTCAGAGATTATCCGCTGGTGGAGTG (SEQ ID NO.22); U3 expression box amplification primer U3box_F: GAGAGCTACCTTACATCAATATGGCGCGCAGATNOGTAGCGGTACATG (SEQ.2) U3box_R: GGTACTATGGCTTAGATGGAATACCCCGTATTCTCTGCCCTTGTTGC (SEQ ID NO. 24); downstream fragment amplification primer c1_downF: GGGTATTCCATCTAAGCCATAGTACCGCGCCTCGAATGACTTGATGAC (SEQ ID NO. 25) and c1_downR: GCCCTACGAGAATGTCGGTTC (SEQ ID NO. 26). The final concentration of the knockout fragmen...
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